CN110031626A - 一种用于胃肠道间质肿瘤迁移的标志物ack1及其应用 - Google Patents
一种用于胃肠道间质肿瘤迁移的标志物ack1及其应用 Download PDFInfo
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Abstract
一种用于胃肠道间质瘤(Gastrointestinal stromal tumors,GIST)肿瘤诊断与迁移的标志物ACK1，该标志物有以下两个特征：（1）与癌旁正常组织相比，ACK1在GIST细胞中高表达且激活；（2）靶向抑制ACK1能抑制GIST细胞的迁移，ACK1是一种非受体酪氨酸激酶，位于细胞质，主要传导跨膜蛋白接收的胞外信号到下游通路,ACK1基因位于3号染色体29，其编码蛋白含1091个氨基酸残基，分子量为145 kDa，ACK1在胃癌、肺癌等常见肿瘤中的高表达以及对肿瘤生长、迁移和浸润等方面所起的重要作用，AIM‑100是ACK1特异性靶向抑制剂。
Description
技术领域
本发明涉及作用于GIST肿瘤迁移的标志物，属于生物医学分析领域。
背景技术
胃肠道间质瘤(Gastrointestinal stromal tumors, GIST)是最常见的胃肠道间质肿瘤，每年每百万人群中就有6.8个人被检查出患有GIST，患者为中老年居多，还有不到1%的GISTs年龄在20岁以下。GIST 通常原发于胃部（68%）和小肠（25-30%）。受体酪氨酸激酶（Receptor tyrosine kinase, RTK）KIT与血小板源性生长因子受体α多肽（platelet—derived growth factor receptor alpha，PDGFRA）基因突变是GIST发生与转移的主要因素。研究证明， GISTs有85%KIT突变或10-15%PDGFRA基因突变。还有10-15%不存在KIT/ PDGFRA突变的GISTs，常称之为“wild type” GIST。目前，GIST的主要诊断标志物包括KIT（CD117）、PKCθ（PRKCQ）、DOG1、CAII四个。
GIST在近几十年经历了单纯性的手术治疗→靶向治疗→综合治疗→个体化治疗这一系列的快速发展。手术切除治疗复发率高，放化疗更是弊大于利，因此酪氨酸激酶抑制剂TKI是许多GIST患者的最佳选择，伊马替尼（Imatinib, IM）是治疗GIST的首选药物。由于KIT与PDGFRA基因在GIST内高频率突变，IM对于大多数GIST患者治疗效果良好，但在用药2-3年后均不同程度地出现了耐药现象。
ACK1（Activated cdc42-associated kinase1）是一种非受体酪氨酸激酶，位于细胞质，主要传导跨膜蛋白(RTK)接收的胞外信号到下游通路。ACK1基因位于3号染色体29，其编码蛋白含1091个氨基酸残基，分子量为145 kDa。ACK1在胃癌、肺癌等常见肿瘤中的高表达以及对肿瘤生长、迁移和浸润等方面所起的重要作用。AIM-100是ACK1特异性靶向抑制剂。
发明内容
本发明的目的在于为GIST诊断和抑制迁移寻找新的标志物。其方法目的是通过如下技术方案来完成的，一种用于胃肠道间质肿瘤迁移的标志物ACK1及其应用，所述该标志物有以下两个特征：第一，它与癌旁正常组织相比，ACK1在GIST细胞中高表达且激活；第二：靶向抑制ACK1能抑制GIST细胞的迁移。
作为优选：检测ACK1在GIST细胞中高表达且激活的步骤为：
a.将肿瘤组织和癌旁组织破碎；细胞用预冷的PBS清洗一遍；加入蛋白裂解缓冲液收集裂解液转移至1.5 mLEp管中；放于4℃冰箱的转盘上旋转过夜；13,000 rpm、4℃离心30min； 将上清液移至新离心管中，记录体积。测量蛋白浓度，定量至2 μg/μL并与LoadingBuffer混匀。将蛋白样品放入100℃金属浴煮沸10 min，离心，SDS-PAGE凝胶电泳;westernblot,ECL显色液A液和B液按1:1混合后滴到膜上，用超灵敏化学发光成像仪成像；
b.免疫共沉淀：裂解细胞，蛋白定量如步骤a；加入琼脂糖珠4℃混合30min，取上
清加入抗体4℃旋转混合2h，4℃离心3 min，将琼脂糖珠离心至管底；将上清小心吸去，琼脂糖珠用裂解缓冲液洗3次；加20 μL loading buffer ，沸水煮10分钟； SDS-PAGE，western blot。
作为优选：靶向抑制ACK1能抑制GIST细胞的迁移的检测包括如下步骤：
a.伤口愈合实验：接种细胞至六孔板里，放置细胞培养箱中培养过夜；第二天用100
μL枪头垂直划三条竖线；用PBS漂洗2次，加入新鲜含药物的培养基培养；选取固定时间点进行拍照记录，直至对照组伤口愈合。
b. 基质胶transwel细胞侵袭实验：预冷24孔板与小室，将无血清培养基稀释的基质胶铺于小室；待基质胶凝固接种细胞于小室，小室内为含药无血清培养基，孔内为含药正常培养基；48h后吸去小室内培养基，PBS清洗小室，加入甲醇固定细胞晾干；加入结晶紫将细胞染色，拍照。
ACK1是一种非受体酪氨酸激酶，位于细胞质，主要传导跨膜蛋白接收的胞外信号到下游通路。ACK1基因位于3号染色体29，其编码蛋白含1091个氨基酸残基，分子量为145kDa。ACK1在胃癌、肺癌等常见肿瘤中的高表达以及对肿瘤生长、迁移和浸润等方面所起的重要作用。AIM-100是ACK1特异性靶向抑制剂。
附图说明
图1是ACK1在GIST肿瘤细胞株的表达，
图2是ACK1在GIST肿瘤组织和癌旁组织中的表达，
图3是ACK1与KIT免疫共沉淀，PY99染色结果，
图4是ACK1靶向抑制对GIST-T1细胞迁移的影响，
图5是ACK1靶向抑制对 GIST430细胞迁移的影响，
图6是ACK1靶向抑制对 GIST-T1细胞侵袭的影响，
图7是ACK1靶向抑制对 GIST430细胞侵袭的影响，
图8是ACK1靶向抑制对 GIST-T1、430细胞侵袭定量分析结果。
具体实施方式
下面将结合具体实施例以及附图对本发明作详细介绍，本发明在实施中选用的生产设备都是本领域的常用设备，应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
一种用于GIST肿瘤诊断与迁移的标志物，其特征在于所述两个方面的内容中，分别包含有如下步骤：
第（1）方面的内容中包括如下三个步骤：
a. 将GIST细胞裂解，western blot；
b. 将肿瘤组织和癌旁组织裂解，western blot；
c. ACK1与KIT免疫共沉淀，PY99染色；
第（2）方面的内容中包括如下两个步骤：
d. 将GIST细胞接种至六孔板中，第二天，用10μL 枪头垂直划线，使用含不同浓度药物培养基培养，每天定点拍照，直至对照组伤口愈合；
e. 将GIST-T1、GIST430基质胶 transwell细胞侵袭实验。
本发明的要点是首先通过western blot检测ACK1在细胞株、肿瘤样本及癌旁组织中的表达，再通过ACK1与KIT免疫共沉淀检测ACK1的激活，最后通过伤口愈合实验和基质胶细胞侵袭实验证明单靶向抑制ACK1可以抑制GIST细胞迁移；具体包括以下步骤：
a. 肿瘤组织和癌旁组织破碎；细胞用预冷的PBS清洗一遍；加入蛋白裂解缓冲液；收
集裂解液转移至1.5 mLEp管中；放于4℃冰箱的转盘上旋转过夜；13,000 rpm、4℃离心30 min； 将上清液移至新离心管中，记录体积。测量蛋白浓度，定量至2 μg/μL并与LoadingBuffer混匀。将蛋白样品放入100℃金属浴煮沸10 min，离心，SDS-PAGE凝胶电泳;westernblot,ECL显色液A液和B液按1:1混合后滴到膜上，用超灵敏化学发光成像仪成像。细胞株显影结果如图1，GIST细胞株包括GIST-T1、GIST882、GIST48、GIST430、GIST62、GIST522、GIST48B、GIST430B，其中，GIST-T1、GIAT882为KIT一次点突变，是IM敏感型细胞株；GIST48、GIST430为KIT两次点突变，是IM耐药性细胞株；GIST62、GIST522、GIST48B、GIST430B 为KIT表达丢失细胞株，对IM抗药。肿瘤组织和癌旁组织显影结果如图2，其中N为癌旁组织，T为肿瘤组织。本实验重复三次。
结果表明ACK1在GIST细胞株和肿瘤组织中高表达，在癌旁组织中不表达。
b.免疫共沉淀：裂解细胞，蛋白定量如步骤a；加入琼脂糖珠4℃混合30min，取上
清加入抗体4℃旋转混合2h，4℃离心3 min，将琼脂糖珠离心至管底；将上清小心吸去，琼脂糖珠用裂解缓冲液洗3次；加20 μL loading buffer ，沸水煮10分钟； SDS-PAGE，western blot，结果如图3。本实验重复两次。
结果表明，ACK1以GIST细胞中磷酸化形式激活。
c. 伤口愈合实验：接种细胞至六孔板里，放置细胞培养箱中培养过夜；第二天用100
μL枪头垂直划三条竖线；用PBS漂洗2次，加入新鲜含药物的培养基培养；选取固定时间点进行拍照记录，直至对照组伤口愈合，结果如图4、图5。本实验重复3次。
结果表明，在IM敏感型细胞株GIST-T1和IM耐药性细胞株GIST430中，靶向抑制ACK1均能抑制GIST细胞的迁移。
d.基质胶transwel细胞侵袭实验：预冷24孔板与小室，将无血清培养基稀释的基质
胶铺于小室；待基质胶凝固接种细胞于小室，小室内为含药无血清培养基，孔内为含药正常培养基；48h后吸去小室内培养基，PBS清洗小室，加入甲醇固定细胞晾干；加入结晶紫将细胞染色，拍照，结果如图6、图7；用醋酸将染色细胞洗脱下来测OD值分析，结果如图8。本实验重复3次。
结果表明，单靶向抑制ACK1能抑制GIST细胞侵袭。
Claims (3)
1.一种用于胃肠道间质肿瘤迁移的标志物ACK1及其应用，其特征在于所述该标志物有以下两个特征：第一，它与癌旁正常组织相比，ACK1在GIST细胞中高表达且激活；第二：靶向抑制ACK1能抑制GIST细胞的迁移。
2.根据权利要求1所述的用于GIST肿瘤诊断与迁移的标志物ACK1，其特征在于所述检测ACK1在GIST细胞中高表达且激活的步骤为：
a.将肿瘤组织和癌旁组织破碎；细胞用预冷的PBS清洗一遍；加入蛋白裂解缓冲液收集裂解液转移至1.5 mLEp管中；放于4℃冰箱的转盘上旋转过夜；13,000 rpm、4℃离心30min； 将上清液移至新离心管中，记录体积;
测量蛋白浓度，定量至2 μg/μL并与Loading Buffer混匀;将蛋白样品放入100℃金属浴煮沸10 min，离心，SDS-PAGE凝胶电泳;western blot,ECL显色液A液和B液按1:1混合后滴到膜上，用超灵敏化学发光成像仪成像；
b.免疫共沉淀：裂解细胞，蛋白定量如步骤a；加入琼脂糖珠4℃混合30min，取上
清加入抗体4℃旋转混合2h，4℃离心3 min，将琼脂糖珠离心至管底；将上清小心吸去，琼脂糖珠用裂解缓冲液洗3次；加20 μL loading buffer ，沸水煮10分钟； SDS-PAGE，western blot。
3.根据权利要求1所述的用于GIST肿瘤诊断与迁移的标志物ACK1，其特征在于靶向抑制ACK1能抑制GIST细胞的迁移的检测包括如下步骤：
a.伤口愈合实验：接种细胞至六孔板里，放置细胞培养箱中培养过夜；第二天用100
μL枪头垂直划三条竖线；用PBS漂洗2次，加入新鲜含药物的培养基培养；选取固定时间点进行拍照记录，直至对照组伤口愈合;
b. 基质胶transwel细胞侵袭实验：预冷24孔板与小室，将无血清培养基稀释的基质胶铺于小室；待基质胶凝固接种细胞于小室，小室内为含药无血清培养基，孔内为含药正常培养基；48h后吸去小室内培养基，PBS清洗小室，加入甲醇固定细胞晾干；加入结晶紫将细胞染色，拍照。
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